Basolateral Na-K-CI cotransport (NKCC) in lining epithelial cells of the lung is crucial for optimal mucociliary clearance because it supplies CI for fluid secretion. Disease states that disrupt CI and fluid movement, such as asthma, chronic bronchitis, and cystic fibrosis (CF), and exposure to foreign irritants contribute to excess mucus and abnormal mucocillary clearance. Treatments aimed at correcting deficient CI secretion might fail to activate NKCC or might compromise NKCC activity thus preventing CI secretion and ultimately failing to correct excess mucus accumulation. Hence, in this proposal, we pose the question: how is NKCC turned on to support transepithelial CI secretion? New evidence from this laboratory points to protein kinase C (PKC)- dependent activation of NKCC in tracheal epithelial cells (TEC) by hormone (alpha1-adrenergic (AR)) stimulation and by hyperosmotic stress. However, modulation of NKCC is more complicated than activation of PKC. Although, intracellular CI levels (CIi) are depicted in models of NKCC to directly modulate CI flux across a plasma membrane through a CI electrochemical gradient, in TEC, stimuli that are expected to increase CIi activate NKCC. Preliminary studies strongly implicate an intracellular signalling pathway as a focal point for CI-dependent activation of NKCC. This will be studied in detail in the following specific aims: 1) To test the hypothesis that CIi modulates NKCC activation by alpha1-AR agonist or by hyperosmotic stress. Changes in [CI]i induced by the two stimuli will be determined. CIi levels will be manipulated to study subsequent effects on activation of NKCC and on NKCC activity. The kinetics of NKCC deactivation will be investigated. 2) To test the hypothesis that CIi modulates activity of protein kinases and phosphatases required for modulation of NKCC activity. Altered activity of protein kinases and phosphatases and the CI-dependence of the enzyme activities will be studied using specific substrates, including NKCC from human and kidney. stPP isotype(s) required for deactivation of NKCC will be identified. 3) To test the hypothesis that CIi modulates protein-protein interactions between NKCC and PKC and/or protein phosphatases. Protein-protein interaction and its dependence on [CI] and activated enzyme will be investigated using immunopurified NKCC, PKC, and protein phosphatases. Shifts in in vivo localization of enzymes with stimulation will be determined by confocal microscopy. A long term outcome of this project is the development of pharmacological tools to manipulate protein-protein interactions that facilitate association of PKC and NKCC to modulate NKCC activity in pathophysiological states.